The present invention relates to certain sugars. More specifically, this invention provides glycosylated polyamine compounds, methods for their synthesis, characterization, their use as ligands in preparing metal complexes for developing analytical methodology, and their pharmaceutical uses, for example, as antitumor agents.
Glycosylated amines, also variously known as N-glycosides, glycosylamines, or aminoglycosides, are formed by reacting a carbonyl containing sugar molecule with an amine. Glycosylated amines are known in the fields of polymer chemistry, and cosmetics. For example, glycosylated amines from primary amines of intermediate molecular weight have been reported to be good wetting agents. Mitts, E. and Hixon, R. M., J. Am. Chem. Soc., 66: 483 (1944). Glycosylated amines as a class have been reported as components for detergents and cosmetics, surfactants, polymers, sweeteners and as liquid crystalline compounds. Lammers, et al., Tetrahedron, 59: 8103 (1994). Glycosylamines also have been used in kraft pulping liquor in the wood processing industry. MacLeod, J. M., Carbohydrate Res., 75: 71 (1979).
Glycosylated amines also play a vital role at the cellular level, because they are essential components of nucleic acids, wherein the ring nitrogen atoms of purine or pyrimidine bases form N-glycosyl linkages with carbon atom 1 of D-ribose or 2-deoxy-D-ribose, which are incorporated into ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), respectively. While the physiological functions of some of the glycosylated amines appear to have been examined, their potential as pharmacological agents has not been fully explored.
Lammers et al. supra, have disclosed the preparation of mono- and di-glycosylamines, wherein the amine and the saccharide were mixed in water under reductive amination conditions. However the yields were poor. Mitts et al. supra, reported the preparation of N,Nxe2x80x2-propylenediglucamine by refluxing glucose with propylenediamine in methanol followed by reduction over activated Raney Nickel and at high pressures and temperatures. The yield appeared to be very low in this case also because it was reported that only a very small amount of the reduced compound was isolated. Mitts et al. further disclosed that attempts to isolate and characterize the condensation products of amines such as isopropylamine, 2-aminooctane, and propylenediamine with glucose were not successful. Accordingly, there exists a need for a synthetic procedure to prepare glycosylated amines and glycosylated polyamines comprising a variety of sugars and amines.
Many analytical techniques have been developed to characterize glycosylamines. Only recently, however, has the research focussed on investigation of linkage information of metal cationized oligosaccharides by mass spectrometry (MS) and tandem mass spectrometry (MSxe2x80x3). Asam, M. R. and Glish, G. L., J. Am. Soc. Mass Spectrom., 8: 987 (1998); Weiskopf, et al. Rapid Com. Mass Spec., 11: 1493 (1997); Hofmeister, et al., J. Am. Chem. Soc., 113: 5964 (1991); and Fura, A. and Leary, J. A., Anal. Chem., 65: 2805 (1993).
Mass spectrometry is not a tool traditionally used to distinguish stereoisomers. However, the stereochemistry of individual monosaccharides as well as a versus xcex2 configuration of glycosidic bonds in disaccharides can be determined by MSxe2x80x3. See for example, Gaucher, S. P. and Leary, J. A., Anal. Chem., 70: 3009 (1998); Smith, et al. J. Org. Chem., 62: 2152 (1997). This method involves cationizing the saccharide using a metal-ligand system such as Zn(diethylenetriamine)2Cl2 or Ni(1,3-diaminopropane)3Cl2, by allowing metal N-glycoside complexes to form in solution. Yano, S., Coord. Chem. Rev., 92: 113 (1988); and Yano, et al. J. Chem. Soc., Dalton Trans., 1699 (1993). The complexes are then transferred from solution to the gas phase by electrospray ionization (ESI) or fast atom bombardment (FAB) and analyzed by tandem mass spectrometry (MS/MS). See for example, Gaucher and Leary, supra; Smith and Leary, supra; and Smith, et al., supra. The axial versus equatorial stereochemistry of the C2 and C4 hydroxyl groups could be differentiated by the cross ring cleavage patterns observed in the gas phase.
In the above-described mass spectrometric procedures, it is time consuming to screen the efficacy of different metals for a given saccharide or ligand because each individual metal-ligand complex requires synthesis a priori. Accordingly, methods for rapid synthesis of metal-ligand complexes are needed so that the metal-ligand complexes so formed can be readily analyzed upon their synthesis.
All literature references, patents, and patent applications cited in this specification are hereby incorporated by reference in their entirety.
The present invention discloses glycosylated polyamines, methods for their preparation and use, and pharmaceutically acceptable compositions comprising gylcosylated polyamines. Glycosylated polyamines, in one embodiment, are prepared from two or more saccharides and a polyamine. In one embodiment, the glycosylated polyamine compound has the following formula (Formula I):
R1xe2x80x94Zxe2x80x94R2xe2x80x83xe2x80x83(Formula I)
wherein: each of R1 and R2 is independently a monosaccharide residue or an oligosaccharide residue; Z is an aliphatic polyamino linker that is the residue of an aliphatic polyamine comprising at least two amino groups, each of which is independently a primary or secondary amino group; and each of R1 and R2 is linked through its anomeric carbon at its 1 position to a different amino group of the aliphatic polyamino linker to form a glycosidic bond;
provided that when each of R1 and R2 is the same and is a glucose, galactose, mannose, or cellobiose residue, Z is the residue of an aliphatic polyamine other than ethylenediamine or diaminopropane;
and pharmaceutically acceptable salts, prodrugs and derivatives thereof.
In one embodiment, each of R1 and R2 of Formula I is an oligosaccharide residue. In another embodiment, at least one of R1 and R2 has a group other than hydrogen in equatorial conformation at the C2 position that is adjacent to the anomeric carbon atom linked to the aliphatic polyamine.
In a further embodiment, the group in equatorial conformation at C2 position is a hydroxy, alkoxy, halo, lower alkyl, amino, N-acetyl, N-alkyl, N-hydroxy, N-alkoxy, aminothiol, amino alcohol, spermine, or nitro group, and optionally a hydrogen in the axial conformation.
In one embodiment of Formula I, R1 is a monosaccharide residue and R2 is an oligosaccharide residue. In another embodiment, each of R1 and R2 of Formula I is a monosaccharide residue. In yet another embodiment, each of R1 and R2 of Formula I is a hexose residue. In a further embodiment, each of the hexose residues is independently substituted by one or more of the following groups: a lower alkyl, lower alkoxy, acyl, carboxy, carboxyamino, amino, acetamido, halo, thio, or nitro; provided that the anomeric carbon has a free hydroxyl group to form a glycosidic linkage with the aliphatic polyamino linker.
In one embodiment, at least one of R1 and R2 has a group other than hydrogen in equatorial conformation at the C2 position that is adjacent to the anomeric carbon atom linked to the aliphatic polyamine. In another embodiment, the group in equatorial conformation at C2 position is a hydroxy, alkoxy, halo, lower alkyl, amino, N-acetyl, N-alkyl, N-hydroxy, N-alkoxy, or nitro group, and optionally a hydrogen in the axial conformation.
In one embodiment, the aliphatic polyamino linker of Formula I is a residue of diethylenetriamine. In a further embodiment, the diethylenetriamine residue is substituted by one or more the following groups: lower alkyl, hydroxy, lower alkoxy, amino, acyl, acetamido, halo, or nitro; provided that there is at least one amino group per each saccharide residue to form a glycosidic linkage.
Another embodiment presents a compound of Formula I, wherein each of R1 and R2 is the same and is a glucose, galactose, allose or fucose residue and the aliphatic polyamine linker is the residue of diethylenetriamine. Some specific embodiments include: diglucosyl-diethylenetriamine; digalactosyl-diethylenetriamine; diallosyl-diethylenetriamine; and difucosyl-diethylenetriamine; and their pharmaceutically acceptable salts, prodrugs and derivatives. One such salt may be HCl salt.
Pharmaceutically acceptable compositions comprising glycosylated polyamines, such as a compound of Formula I and pharmaceutically acceptable salts, prodrugs and derivatives thereof are also provided.
Glycosylated polyamine compounds, for example, of formula 1, are effective as anticancer agents. Such anticancer activity may include inhibition of tumor cell growth, or multiplication or tumor size. Some examples of such compounds are diglycosylated diethylenetriamines, such as diglucosyl diethylenetriamine, digalactosyl diethylenetriamine, difucosyl diethylenetriamine, and diallosyl diethylenetriamine.
Accordingly, compounds of Formula I can be employed in methods to treat cancers or tumors. Such methods include providing one or more compounds of Formula I, their pharmaceutically effective salts, prodrugs and derivatives in an effective amount to treat a cancer or reduce the tumor cell growth or multiplication or tumor size. Exemplary cancers that may be treated include: leukemia, non-small-cell lung cancer, small-cell lung cancer, colon cancer, a cancer of the central nervous system, melanoma, ovarian cancer, breast cancer, renal and prostate cancer. The above-described compounds of Formula I can also be used in preparing one or medicaments to treat one or more of such cancers.
One specific embodiment of Formula I is diglucosyl diethylenetriamine. The diglucosyl diethylenetriamine can be prepared, for example, in a salt form such as HCl salt, which has the structure: 
Yet another specific embodiment of Formula I is digalactosyl diethylenetriamine. The digalactosyl diethylenetriamine can be prepared, for example, in a salt form, such as HCl salt, which has the structure: 
Another specific embodiment of Formula I is difucosyl diethylenetriamine. The difucosyl diethylenetriamine can be prepared, for example, in a salt form, such as HCl salt, which has the structure: 
Another specific embodiment of Formula I is diallosyl diethylenetriamine. The diallosyl diethylenetriamine can be prepared, for example, in a salt form such as HCl salt, which has the structure: 
The present invention also provides a metal-polyamine-N-glycosyl complex of Formula II:
[R1xe2x80x94Zxe2x80x94R2]. Yxe2x80x83xe2x80x83(Formula II)
wherein in one embodiment, [R1xe2x80x94Zxe2x80x94R2] is represented by Formula I; and Y is a metal compound; and pharmaceutically acceptable salts, prodrugs and derivatives thereof.
In one embodiment, each of R1 and R2 is a hexose residue. In yet another embodiment, each of R1 and R2 is the same and is a glucose, galactose, allose or fucose residue.
In one embodiment of Formula II, Z is an aliphatic polyamino linker which is a residue of a polyamine selected from the group consisting of ethylene diamine, propylene diamine, diethylene triamine. In an embodiment of Formula II, the metal compound Y is a metal such as zinc, or a metal salt such as zinc chloride, zinc acetate, zinc triflate, sodium chloride magnesium chloride, copper chloride, cobalt chloride, nickel chloride, or calcium carbonate. In addition, the metal salts can be also those of organic origin such as sulfonate triflate or tosylate.
An embodiment of a glycosylated polyamine-zinc complex is N,Nxe2x80x2-dihexosyl-diethylenetriamine-zinc chloride which has the following structure: 
In one embodiment, a method for preparing a metal-polyamine-N-glycosyl complex, for example, of Formula II:
[R1xe2x80x94Zxe2x80x94R2]. Yxe2x80x83xe2x80x83Formula II
is provided which comprises:
a) providing a glycosylated polyamine compound, for example, of Formula I, [Rxe2x80x94Zxe2x80x94R2]; b) reacting the compound of Formula I with a metal compound, Y, and a salt, such as ammonium hydroxide, in a solvent, such as methanol, to form a metal complex having the formula, Formula II, [R1xe2x80x94Zxe2x80x94R2]. Y; and, optionally, c) isolating the metal complex obtained in step b).
The metal compound Y and the compound of Formula I are preferably reacted in about equimolar amounts.
In one embodiment, the metal compound comprises a metal, such as zinc, or a metal salt, such as zinc chloride. An exemplary general reaction scheme to prepare a zinc chloride-diethylenetriamine-dihexosyl complex is shown below: 
The present invention also provides an analytical method to determine the stereospecificity of a glycosidic linkage between two sugars in a disaccharide. The method comprises the steps of: a) providing a glycosylated polyamine, for example, of Formula I, wherein at least one of R1 and R2 is a disaccharide; b) cationizing the glycosylated polyamine using a metal compound Y to form the corresponding metal-polyamine-N-glycoside complex of Formula II; c) ionizing the metal-polyamine-N-glycoside complex; and d) detecting the ions characteristic of a particular stereospecific linkage of the disaccharide using one or more mass spectrometers.
In one embodiment, the metal compound Y comprises zinc chloride or nickel chloride. In another embodiment, the metal-polyamine-glycoside complex is ionized through an electrospray ionization or a fast atom bombardment ionization technique. In yet another embodiment, the ion detection is accomplished by using two or more mass spectrometers arranged in tandem, in space and time, for example as triple quad instruments or those employing ion trap or ion cyclotron resonance technology.
The present invention also discloses a method for detecting the presence of an axial or equatorial conformation of a group, for example, at the C2 position of a saccharide, which method comprises the steps of: a) reacting an aliphatic polyamine and the saccharide in the presence of a precipitating agent; b) observing for a precipitate in the reaction mixture within a certain time, such as from within a few minutes to within a few hours, for example, 3-8 hours: and c) noting the presence of equatorial conformation of the group at the C2 position if a precipitate is observed in step b).
In one embodiment, the aliphatic polyamine and the saccharide are reacted at a certain ratio, for example, at about a 1:2 molar ratio. In another embodiment, the precipitating agent is present in about 1 molar concentration. In some embodiments, the precipitate can be observed within a few minutes, whereas in certain other embodiments, the precipitate is formed within a few hours; provided that the saccharide has an equatorial substitution at the C2 position.
In one embodiment, the substituent at the C2 position is a hydroxyl group. Other examples of substituents at the C2 position include alkoxy, halo, lower alkyl, amino, substituted amino, and nitro groups. The saccharide may be for example, a monosaccharide such as a pentose, or hexose, or an oligosaccharide. The precipitating agent can be any acidic salt that provides a halo counterion, for example, HCl, HBr, HI. Preferably, the counterion is a chloro ion. The aliphatic polyamine includes polyamines such as diethylene triamine, triethylenetetramine, ethylene diamine, and diaminopropane.
The present invention provides glycosylated polyamines, methods for their synthesis and methods of using them, for example, as anticancer agents. Metal complexes of glycosylated polyamines, the preparation of such metal complexes, and analytical methods using the metal complexes are also provided. Methods for detecting equatorial and axial conformations of a group other than hydrogen at the C2 position of a saccharide molecule also are provided.
Glycosylated Polyamine Compounds
This invention provides a compound of the formula (Formula I):
R1xe2x80x94Zxe2x80x94R2xe2x80x83xe2x80x83(Formula I)
wherein in one aspect, each of R1 and R2 is independently a saccharide residue, such as a monosaccharide residue or an oligosaccharide residue; Z is a polyamino linker that is the residue of a polyamine comprising at least two amino groups, each of which is independently a primary or secondary amino group; and each of R1 and R2 is linked through its anomeric carbon at its 1 position to a different amino group of the polyamino linker to form a glycosidic bond; provided that when both R1 and R2 are the same and represent glucose, galactose, mannose, or cellobiose residues, Z is a residue of a polyamine other than ethylenediamine or diaminopropane; and pharmaceutically acceptable salts, prodrugs and derivatives thereof.
In Formula I, each of R1 and R2 can independently represent the same saccharide residue or residues of different saccharides. For example, R1 and R2 can both be monosaccharide residues or R1 can be a monosaccharide residue and R2 can be an oligosaccharide residue, or vice versa. Further, each of R1 and R2 can be independently a monosaccharide residue such as a pentose, hexose or heptose residue. Thus, R1 can be a pentose residue and R2 can be a hexose residue.
The term xe2x80x9csaccharide residuexe2x80x9d as used herein, refers to the portion of a saccharide that is without a hydroxyl group at the reducing end (C1) of the saccharide. The saccharide residue is thus the portion of the saccharide attached to the polyamine. Such structures may also be referred to as saccharyl. Thus, for example, the phrases xe2x80x9cglucose residuexe2x80x9d and xe2x80x9cglucosylxe2x80x9d refer to the same glucose structure which lacks a hydroxyl group at its reducing end (C1). Similarly, xe2x80x9cpolyamine residuexe2x80x9d refers to the polyamino portion of the glycosylated polyamine that is bonded to various saccharide residues. These terms are readily understood by one of ordinary skill in the art. This terminology can also be illustrated from the following structure of compound 1, wherein each of R1 and R2 represents, for example, glucosyl or a glucose residue and Z represents a polyamine residue: 
Exemplary monosaccharide residues include residues of a pentose, hexose, or a heptose. Non-limiting examples of pentoses include arabinose, ribose, ribulose, xylose, lyxose, and xylulose. Non-limiting examples of hexoses include glucose, galactose, fructose, fucose, mannose, allose, altrose, talose, idose, psicose, sorbose, and tagatose. Non-limiting examples of heptoses include mannoheptulose and sedoheptulose.
Some saccharide residues may have a hydroxyl group at the C2 position in an axial or equatorial conformation. For example, glucosyl residue has a hydroxyl group in equatorial conformation at its C2 position, whereas a mannosyl residue, for example, has a hydroxyl group in axial conformation at its C2 position.
In some cases, the saccharide residue may possess a group other than a hydrogen or hydroxyl at its C2 position. Some examples of groups that may be present at the C2 position include groups such as alkoxy, halo, lower alkyl, amino, N-acetyl, N-alkyl, N-hydroxy, N-alkoxy, aminothiol, amino alcohol, spermine, or nitro group. Such groups may be present in equatorial or axial conformation at the C2 position.
The saccharides of the present invention may also be substituted with various groups. Such substitutions may include lower alkyl, lower alkoxy, acyl, carboxy, carboxyamino, amino, acetamido, halo, thio, nitro, keto, and phosphatyl groups, wherein the substitution may be at one or more positions on the saccharide, except for the anomeric carbons which form the glycosidic bond. Moreover, the saccharides may also be present as a deoxy saccharide. Examples of such substituted saccharides include: D-glucosamine and D-galactosamine, which are 2-amino-2-deoxy glucose and 2-amino-2-deoxy galactose, respectively. Examples of carboxy-containing saccharides include aldonic, aldaric, and uronic acids. Examples of carboxy-containing amino sugars include N-acetylmuramic acid and N-acetylneuraminic acid, wherein each is a six-carbon amino sugar linked to a three-carbon sugar acid.
Preparation of substituted sugars with the above-listed substituents is well within the ordinary skill in the art. General methods for preparing substituted sugars have been described in, for example, U.S. Pat. No. 5,874,413; Kissman et al., xe2x80x9c5-deoxy-5-fluoro-D-ribofuranosyl derivatives of certain purines, pyrimidines and 5,6-dimethylbenzimidazolexe2x80x9d J. Chem. Soc. (1958); 80:5559-5564; Kissman et al., xe2x80x9cThe synthesis of certain 5-deoxy-D-ribofuranosylpurinesxe2x80x9d J. Am. Chem. Soc. (1957) 79:5534-5540; Weiss et al., xe2x80x9cThe reaction of periodate with aminosugars. Anomalous overoxidations of aminofuranosidesxe2x80x9d J. Am. Med. Soc. (1959) 81:4050-4054; Verheyden et al., xe2x80x9cHalo sugar nucleosides. IV. Synthesis of some 4xe2x80x25xe2x80x2-unsaturated pyrimidine nucleosidesxe2x80x9d J. Org. Chem. (1974); 39:3573-3579.
Saccharides exist as stereoisomers, optical isomers, anomers, and epimers. The meaning of saccharide as used herein encompasses such isomers, anomers and epimers. Thus, a hexose for example can be either an aldose or a ketose, and can be of D- or L-configuration, can assume either an alpha or beta conformation, and can be a dextro- or levo-rotatory with respect to plane-polarized light.
An oligosaccharide includes two or more monosaccharides joined through glycosidic linkage. An oligosaccharide may result from a glycosidic linkage between, for example, a hexose and another hexose or between a pentose and a hexose. An oligosaccharide of this invention may comprise of 2-6 saccharyl units, preferably, 2-4 saccharyl units, and more preferably, 2-3 saccharyl units.
Nonlimiting examples of oligosaccharides include lactose, maltose, cellobiose, gentiobiose, melibiose, isomaltose, mannobiose and xylobiose. The oligosaccharides have a free anomeric carbon to form glycosidic linkage with the polyamine. The preparation of several oligosaccharides is well-known in the art. They can be obtained by partial hydrolysis of polysaccharides or synthesized from the desired monosaccharides. See for example, Morrison and Boyd, Organic Chemistry, Prentice-Hall, Chapter on Carbohydrates, latest edition. In addition, several oligosaccharides can be purchased from commercial sources or can be custom made using ordinary skill in the art.
Oligosaccharides also exist in many isomeric, epimeric and anomeric forms and the oligosaccharides described herein include such isomeric, epimeric and anomeric forms.
In one aspect, each of R1 and R2 of Formula I is an oligosaccharide residue. In some cases, at least one of R1 and R2 has a group other than hydrogen in equatorial conformation at the C2 position that is adjacent to the anomeric carbon atom linked to the aliphatic polyamine. In some other cases, each of R1 and R2 has a group other than hydrogen in equatorial conformation at that C2 position. The group in equatorial conformation at C2 position may be a hydroxy, alkoxy, halo, lower alkyl, amino, N-acetyl, N-alkyl, N-hydroxy, N-alkoxy, aminothiol, amino alcohol, spermine, or nitro group.
In one aspect, in Formula I, R1 is a monosaccharide residue and R2 is an oligosaccharide residue. In another aspect of Formula I, each of R1 and R2 is a monosaccharide residue. In yet another aspect of Formula I, each of R1 and R2 is a hexose residue. Each of the hexose residues may be independently substituted by one or more of the groups: a lower alkyl, lower alkoxy, acyl, carboxy, carboxyamino, amino, acetamido, halo, thio, or nitro; provided that the anomeric carbon has a free hydroxyl group to form a glycosidic linkage with the aliphatic polyamino linker.
In some cases, at least one of R1 and R2 has a group other than hydrogen in equatorial conformation at the C2 position that is adjacent to the anomeric carbon atom linked to the aliphatic polyamine. In some other cases, each of R1 and R2 has a group other than hydrogen in equatorial conformation at that C2 position. The group in equatorial conformation at C2 position may be a hydroxy, alkoxy, halo, lower alkyl, amino, N-acetyl, N-alkyl, N-hydroxy, N-alkoxy, aminothiol, amino alcohol, spermine, or nitro group.
xe2x80x9cAliphatic polyaminexe2x80x9d refers to two or more amino groups separated by one or more carbon atoms representing an aliphatic group, wherein the amino groups can be primary, secondary or tertiary, provided that the amino group that is bonded to the anomeric carbon atom of a mono- or oligo-saccharide is either a primary or a secondary amine. Thus, the term xe2x80x9caliphatic polyamino linkerxe2x80x9d refers to the aliphatic polyamino group that links to one or more saccharides, wherein each saccharide, through its anomeric carbon at the 1 position, forms a glycosidic bond with a primary or secondary amino group of the aliphatic polyamine.
In one aspect, the aliphatic polyamino linker includes two or more amino groups and two or more carbons, for example from 4 to 10, provided that the aliphatic polyamino linker has at least two amino groups, wherein each amino group can independently be a primary or secondary amino group. Preferably, the aliphatic polyamino linker has 2-6 such amino groups, and more preferably, 2-4 such amino groups. Particular examples of aliphatic polyamines include ethylene diamine, diaminopropane, diethylenetriamine and triethylenetetramine.
The aliphatic polyamine may be substituted at one or more positions on the aliphatic portion or on the amino portion, with the proviso that there be at least one primary or secondary amino group per saccharide residue (for example, R1 and R2 as described above) to be conjugated. For example, diethylenetriamine may be substituted by one or more the following groups: lower alkyl, hydroxy, lower alkoxy, amino, acyl, acetamido, halo, or nitro; provided that there is at least one primary or secondary amino group per each N-glycosidic linkage to be formed.
xe2x80x9cAliphaticxe2x80x9d refers to a cyclic, branched, or straight chain group containing only carbon and hydrogen, such as methyl, pentyl, and adamantyl. Aliphatic groups can be unsubstituted, or may be substituted with one or more substituents, e.g., halogen, alkoxy, acyloxy, amino, hydroxyl, mercapto, carboxy, benzyloxy, phenyl, benzyl, or other functionality that may be suitably blocked, if desired, for example with a protecting group. Aliphatic groups can be saturated or unsaturated (e.g., containing xe2x80x94Cxe2x95x90Cxe2x80x94 or xe2x80x94Cxe2x95x90Cxe2x80x94 subunits), at one or several positions. Typically, aliphatic groups will comprise 2 to 12 carbon atoms, preferably 2 to 8, and more preferably 2 to 5 carbon atoms.
The phrase xe2x80x9clower alkylxe2x80x9d refers to alkyls having 1-4 carbon atoms, such as methyl, ethyl, propyl or butyl. The phrase xe2x80x9csubstituted aminoxe2x80x9d refers to a primary amino group wherein each of the hydrogens may be independently replaced by groups such as lower alkyl, hydroxyl, alkoxyl, acyl groups such as xe2x80x94C(O)CH3 or C(O)C2H5.
One specific example of a glycosylated polyamine is diglucosyl-diethylenetriamine, wherein each of R1 and R2 is glucosyl and the polyamino linker is a residue of diethylenetriamine. The diglucosyl-diethylenetriamine can be prepared in a salt form such as a HCl salt. The structure of diglucosyl-diethylenetriamine.HCl (1) is shown below: 
Another example of a glycosylated polyamine is digalactosyl-diethylenetriamine, wherein each of R1 and R2 is galactosyl and the polyamino linker is a residue of diethylenetriamine. The digalactosyldiethylenetriamine can be prepared in a salt form, such as a HCl salt. The structure of digalactosyl-diethylenetriamine.HCl (2) is shown below: 
Another example of a glycosylated polyamine is difucosyl-diethylenetriamine, wherein each of R1 and R2 is fucosyl and the polyamino linker is a residue of diethylenetriamine. The difucosyl-diethylenetriamine can be prepared in a salt form such as a HCl salt. The structure of difucosyl-diethylenetriamine.HCl (3) is shown below: 
Yet another example of a glycosylated polyamine is diallosyl-diethylenetriamine, wherein each of R1 and R2 is allosyl and the polyamino linker is a residue of diethylenetriamine. The diallosyl-diethylenetriamine may be provided in a salt form, such as an HCl salt. The structure of diallosyl-diethylenetriamine.HCl (2) is shown below: 
Diglycosylamines may be characterized by methods available in the art, such as, by 13C-NMR and mass spectra and X-ray crystallography. See for example, Lammers, et al, supra at 8105-8116; Gaucher and Leary, supra; Fura and Leary, supra; and Smith, et al, supra.
Methods of Preparation of Glycosylated Polyamines
The compounds of Formula I can be prepared by: a) combining an inorganic acid such as HCl with an aliphatic polyamine; and b) combining each of the saccharides corresponding to the R1 and R2 saccharide residues with the product obtained in step a) to form a glycosylated polyamine product; and optionally, c) isolating the glycosylated polyamine product of step b). Equimolar amounts of the inorganic acid, the polyamine and each of the saccharides may be used in the method.
The aliphatic polyamine is dissolved in an organic solvent such as methanol and kept at about 0xc2x0 C. to room temperature. HCl is provided in a relatively nonpolar solvent such as diethylether. Step a) comprises adding HCl to the polyamine solution dropwise with stirring. In step b), the product of step a) is warmed up, if necessary, to room temperature, and each saccharide corresponding to the R1 and R2 saccharide residue in a polar solvent such as water is added to the product of step a). The reaction mixture is allowed to stir for at least an hour and the product is collected. Optionally, the reaction mixture is kept at 4xc2x0 C. overnight and yields can be improved.
The isolation step c) also can include collecting the glycosylated polyamine product and drying under vacuum, and, optionally, dissolving the dried product in water, and recrystallizing in an organic solvent, such as a 50:50 mixture of methanol and ethanol.
As described above, each of R1 and R2 can represent a variety of saccharide residues while Z is an aliphatic polyamino linker. Some specific examples prepared by this method include diglucosyl diethylenetriamine, difucosyl diethylenetriamine, digalactosyl diethylenetriamine, and diallosyl diethylenetriamine.
A general reaction scheme for preparing a hexosyl diethylenetriamine, wherein both R1 and R2 are hexosyl, is illustrated below: 
Thus, for example, about 1 mole of an inorganic acid such as HCl is combined with about 1 mole of an aliphatic polyamine. About 1 mole each of the saccharides corresponding to the saccharide residues represented by R1 and R2 is reacted with the above aliphatic polyamine.HCl mixture to form R1xe2x80x94Zxe2x80x94R2 product; which can be isolated.
When the two saccharyl groups (represented by R1 and R2), for example, are the same, such as glucosyl, the molar ratio of the saccharide to the polyamine is about 2:1. However, when the two saccharyl groups are different, i.e., when R1 is, for example, a glucosyl and R2 is, for example, a galactosyl, the ratio of glucose to galactose to polyamine is for example about 1:1:1. Thus, one mole of the polyamine reacts with one mole of each saccharide to form one mole of the glycosyl polyamine.
Applications
Glycosylated polyamine compounds, for example, of formula I, are effective as anticancer agents. Such anticancer activity may include inhibition of tumor cell growth, or multiplication or tumor size. Some examples of such compounds are diglycosylated diethylenetriamines, such as diglucosyl diethylenetriamine, digalactosyl diethylenetriamine, difucosyl diethylenetriamine, and diallosyl diethylenetriamine.
Accordingly, compounds of Formula I can be employed in methods to treat cancers or tumors. Such methods include providing one or more compounds of Formula I, their pharmaceutically effective salts, prodrugs and derivatives in an effective amount to treat a cancer or reduce the tumor cell growth or multiplication or tumor size. Exemplary cancers that may be treated include: leukemia, non-small-cell lung cancer, small-cell lung cancer, colon cancer, a cancer of the central nervous system, melanoma, ovarian cancer, breast cancer, renal and prostate cancer. The above-described compounds of Formula I can also be used in preparing one or medicaments to treat one or more of such cancers.
Methods for assaying compounds for biological activity such as anti-cancer activity are well known in the art. See, for example, Li, L. H. et al., Cancer Res., 39: 4816-4822 (1979)).
The glycosylated polyamine compounds of Formula I can be prepared in a pharmaceutically acceptable form, such as a salt suitable for a particular formulation or route of administration in a suitable dose and dosage form.
The glycosylated polyamine also may be in a prodrug or derivative form, i.e., as salts that are capable of being hydrolyzed or solvated under physiological conditions. Examples of such salts include sodium, potassium, and hemisulfate. The term xe2x80x9cprodrugxe2x80x9d is further intended to include lower hydrocarbon groups capable of being hydrolyzed or solvated under physiological conditions, e.g. methyl, ethyl, and propyl.
Prodrugs and derivatives may be prepared by modifying pharmacologically active molecules to enhance certain pharmaceutical characteristics such as oral absorption, plasma half-life, protein binding, or entry through blood-brain barrier. These modified molecules are in general, though not invariably, inactive by themselves, but release the active molecule in vivo upon being acted on by enzymes or chemicals or physiological conditions in vivo. These prodrugs and derivatives are known in the medicinal art. See for example, Bundgaard et al., J. Med. Chem., 32: 2503-7 (1989).
The glycosylated polyamine compounds of Formula I can be provided in a pharmaceutically acceptable carrier. The pharmaceutical composition includes one or more of the glycosylated polyamines in an effective amount for the treatment of a condition. The term xe2x80x9ceffective amountxe2x80x9d refers to an amount sufficient to effect beneficial or desired pharmacological or clinical results. The amount can be determined based on such factors as the type and severity of symptoms being treated, the weight and/or age of the subject, the previous medical history of the subject, and the selected route for administration of the agent. The determination of appropriate xe2x80x9ceffective amountsxe2x80x9d is within the ordinary skill of the art.
The term xe2x80x9cadministrationxe2x80x9d is intended to include routes of administration which allow the agent (e.g. anticancer agent) to perform its intended function, e.g., reducing the tumor mass or growth. Examples of routes of administration which can be used include injection (subcutaneous, intravenous, parenterally, intraperitoneally, etc.), oral, inhalation, trandsdermal, and rectal. Depending on the route of administration, the agent can be coated with or in a material to protect it from the natural conditions which may detrimentally effect its ability to perform its intended function. The administration of the agent is done at dosages and for periods of time effective to significantly reduce or eliminate tumor mass or growth. Dosage regimes may be adjusted for purposes of improving the therapeutic response of the agent. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
The compounds, or their pharmaceutically acceptable salts, prodrugs and derivatives and combinations thereof described herein can be provided and administered in a pharmaceutically acceptable carrier. The phrase xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d is intended to include substances capable of being co-administered with the agent and which allow the agent to perform its intended function, e.g. reducing tumor mass or growth. Examples of such carriers include solvents, dispersion media, delay agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media and agent compatible with the compound can be used with this invention. The compound compositions further can be co-administered with other agents such as art-recognized protein enhancing agents, differentiating agents, and/or adjuvants.
Thus, a glycosylated polyamine compound or a composition comprising the compound, can be administered in an effective amount to treat a condition, such as cancer, or to obtain a desired clinical result, such as remission or cure of such cancerous condition. The compounds and compositions are administered in vitro, in vivo, or ex vivo, depending on the particular circumstances if the treatment, including the disease, dosage, patient""s age, and health among other factors.
xe2x80x9cIn vitroxe2x80x9d use of a material is defined as a use of a material or compound outside a living human, mammal, or vertebrate, where neither the material nor compound is intended for reintroduction into a living human, mammal, or vertebrate. An example of an in vitro use would be the analysis of components of a blood sample using laboratory equipment. xe2x80x9cIn vivoxe2x80x9d use of a material is defined as introduction of the material into a living human, mammal, or vertebrate.
xe2x80x9cEx vivoxe2x80x9d use of a compound is defined as using a compound for treatment of a biological material outside a living human, mammal, or vertebrate, where that treated biological material is intended for use inside a living human, mammal, or vertebrate. For example, removal of blood from a human, and introduction of a compound into that blood, is defined as an ex vivo use of that compound if the blood is intended for reintroduction into that human or another human. Reintroduction of the human blood into that human or another human would be in vivo use of the blood, as opposed to the ex vivo use of the compound. If the compound is still present in the blood when it is reintroduced into the human, then the compound, in addition to its ex vivo use, is also introduced in vivo.
The glycosylated polyamine compounds described herein and their salts, prodrugs may be used directly by themselves or may be incorporated into a medicament preparation for treating an ailment such as cancer. Thus, depending on the intended mode, the compositions may be in the solid, semi-solid or liquid dosage form, such as, for example, injectables, tablets, suppositories, pills, time-release capsules, powders, liquids, suspensions, or the like, including in unit dosage formulations. The compositions, while including an effective amount of a glycosylated polyamine compound of the present invention, or the pharmaceutically acceptable salt thereof, in addition, may include any conventional pharmaceutical excipients and other medicinal or pharmaceutical drugs or agents, carriers, adjuvants, diluents, etc., as customary in the pharmaceutical sciences.
For solid compositions such excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like may be used. The glycosylated polyamines as described above, may be also formulated as suppositories using, for example, polyalkylene glycols, for example, propylene glycol, as the carrier.
Liquid, particularly injectable compositions can, for example, be prepared by dissolving, dispersing, a glycosylated polyamine of the invention in a pharmaceutical solution such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form the injectable solution or suspension.
If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the other substances such as for example, sodium acetate, triethanolamine oleate, etc.
Parenteral injectable administration is generally used for subcutaneous, intramuscular or intravenous injections and infusions. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions or solid forms suitable for dissolving in liquid prior to injection.
The above preparations can be made into slow-release or sustained-released systems, which provide a constant level of dosage. Such sustained release preparations are well-known in the art. See, for example, U.S. Pat. No. 3,710,795. Actual methods of preparing such dosage forms are known, or will be apparent to those skilled in this art, and are in detail described in Remington""s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17th Edition, 1985.
Any of the above pharmaceutical compositions may contain 0. 1-99% of the glycosylated polyamine. An effective dosage may be in the range of 0.001 to 5000 mg/kg/day, preferably 0.01 to 1000 mg/kg/day, more preferably 0.1 to 100 mg/kg/day. Generally, the upper limit for the drug dose determination is its efficacy balanced with its possible toxicity.
The pharmaceutical compositions of this invention can contain one or more compounds of the Formula I described above and, if desired, can be employed in combination with other therapeutic agents including conventional anti-tumor agents known in the art. Suitable examples of such conventional anti-tumor agents which can be used include adriamycin, cisplatin, colchicine, CCNU, BCNU, Actinomycin D, 5-fluorouracil, thiotepa, cytosinearabinoside, cyclophosphamide, mitomycin C, and the like.
The compounds of the present invention may be susceptible to hydrolysis because they possess Cxe2x80x94N glycosidic linkages and this hydrolysis might reduce their therapeutic efficacy against certain cancer cell lines. Accordingly, one aspect of this invention provides compounds of Formula I having Cxe2x80x94C glycosidic linkage, instead of a Cxe2x80x94N glycosidic linkage. An example of a hexosyldiethylenetriamine having a Cxe2x80x94C glycosidic linkage can be shown as below: 
One method of forming such Cxe2x80x94C linkage is described in Ledl, F. and Schleicher, E., Angew. Chem. Int. Ed. Engl., 29: 565 (1990).
Metal Complexes of Glycosylated Polyamines
Metal complexes of glycosylated polyamines may be formed with metal compounds such as metals or metal salts. The metal complexes can be rapidly prepared and characterized for the stereospecificity of their glycosyl linkages. Such stereochemistry-differentiating systems are useful for elucidating total synthesis of complex molecules such as natural products and also for preparing and identifying biologically more active compounds wherein the activity may reside in a particular stereoisomer.
Thus, a compound of the formula R1xe2x80x94Zxe2x80x94R2 (Formula I), can be complexed to a metal compound Y, to produce a metal-polyamine-glycosyl complex having the following structure (Formula II):
[R1xe2x80x94Zxe2x80x94R2]. Yxe2x80x83xe2x80x83(Formula II)
wherein Formula I is as described above and Y is a metal compound.
The metal compound Y is a metal such as zinc, or a metal salt such as zinc chloride, zinc acetate, zinc triflate, sodium chloride magnesium chloride, copper chloride, cobalt chloride, nickel chloride, or calcium carbonate. In addition, the metal salts can be also those of organic origin such as sulfonate triflate or tosylate.
Thus, a glycosylated polyamine-metal complex such as N,Nxe2x80x2-diglucosyl diethylenetriamine can be complexed to a metal compound such as zinc chloride.
One general structure of a dihexosyldiethylenetriamine-zinc chloride complex is shown below: 
In one preferred example of a dihexosyldiethylenetriamine-zinc chloride complex, the hexose is a glucose. In some cases, the hexose residue possesses a group other than a hydrogen in axial conformation at its C2 position, whereas in other cases, the hexose residue possesses a group other than hydrogen in equatorial conformation at its C2 position. Mannose, for example, possesses a hydroxyl group in axial conformation at its C2 position, whereas glucose, for example, possesses a hydroxyl group in equatorial conformation at its C2 position.
Some additional examples of groups that may be present at the C2 position include groups such as alkoxy, halo, lower alkyl, amino, N-acetyl, N-alkyl, N-hydroxy, N-alkoxy, aminothiol, amino alcohol, spermine, or nitro group, and optionally a hydrogen in the axial conformation, provided that at least one of the residues of R1 and R2 has a group that has an acidic hydrogen at the C2 position of the saccharide residue that forms glycosidic bond with the polyamine to form a hydrogen bond with the metal. For example, if R1 is a glucose, R2 can be a glucose that has its C2 hydroxyl substituted by a nitro or a halo group. Additional examples of such acidic hydrogen containing groups include hydroxyl, thio, acyl, carboxyl, and hydroxyamino.
Preparation of Metal Complexes of Glycosylated Polyamines
One method for preparing the metal complexes of glycosylated polyamines comprises mixing a glycosylated polyamine, for example, a compound of the formula [R1xe2x80x94Zxe2x80x94R2], Formula I, as described hereinabove, with an equimolar amount of a metal compound Y and an inorganic salt prepared from a weak base-strong acid system, such as ammonium hydroxide, in an organic solvent, such as methanol, to form a metal complex, which optionally can be further purified.
The method presented herein is a rapid method to prepare metal complexes, wherein the metal complex is often formed in a relatively short time, for example, within a few minutes of the mixing of the glycosylated polyamine and the metal compound as described above. Moreover, the metal complex formed by this method gives rise to a suitable parent ion when analyzed using a mass spectrometer. This parent ion can be collisionally activated to give rise to a product ion that is characteristic of a specific configuration of the glycoside. Thus, the mass spectrum reveals whether the glycosidic linkage is an alpha or a beta linkage.
An exemplary general reaction scheme to prepare a dihexosyl diethylenetriamine zinc chloride complex is shown below: 
Analytical Methods Using Metal Complexes of Glycosylated Polyamines
It is well known that saccharides exist in many stereoisomeric forms. One particular type of such stereoisomerism is the presence of an alpha or beta linkage at the anomeric carbon, giving rise to alpha and beta anomers. A rapid analytical method to distinguish stereoisomers of a disaccharide is provided.
In one aspect, the glycosylated polyamine of Formula I as described above is cationized using a metal compound Y to form a metal-polyamine-N-glycoside complex (Formula II, supra), wherein, at least of one of R1 and R2 is a disaccharide. Some examples of such metal-polyamine-glycosyl complexes include zinc(diethylenetriamine-diglucosyl)2Cl2 or nickel(1,3-diaminopropane-diglucosyl)3Cl2. The metal-polyamine-N-glycoside complex is then ionized and the ions are detected using one or more mass spectrometers.
The metal-N-glycoside complex can be ionized by using a variety of ionization techniques. Some examples of such ionization techniques include, electron impact, chemical ionization, field desorption, electrospray or fast atom bombardment ionizations and matrix assisted laser desorption ionization. The ion detection is accomplished for example, by using two or more mass spectrometers arranged in tandem, in space and time, for example as triple quad instruments or those employing ion trap or ion cyclotron resonance technology. The mass spectrometers can be of different types, and include quadruple mass spectrometers. Further, the mass spectrometers can be operated manually, or automated through a computer program.
Methods for Detecting Axial or Equatorial Conformation
Axial or equatorial conformations are present in many types of structures, including saccharides. The detection of such conformations are of great interest in carbohydrate chemistry because knowledge of the conformation helps in elucidating or establishing total synthesis and also in correlating stereochemistry with biological activity.
The presence of an axial or equatorial conformation of a group other than hydrogen at, for example, the C2 position of a saccharide can be detected by : a) reacting an aliphatic polyamine and the saccharide in the presence of a precipitating agent; b) observing for a precipitate in the reaction mixture; and c) noting the presence of an axial conformation of the group at the C2 position if no precipitate is observed within a certain time after the reaction according to step a).
The aliphatic polyamine and the saccharide are reacted at a certain ratio, for example, at about a 1:2 molar ratio. The precipitating agent is present in less than or equal to 1 molar concentration. In some cases, the precipitate can be observed within a few minutes, whereas in other cases the precipitate is formed within a few hours, provided that the saccharide has a group other than a hydrogen in equatorial conformation at the C2 position.
The group whose conformation at C2 is being determined can be an alkoxy, halo, lower alkyl, amino, N-acetyl, N-alkyl, N-hydroxy, N-alkoxy, aminothiol, amino alcohol, spermine, or nitro group. The saccharide may be, for example, a monosaccharide comprising a pentose, hexose, or an oligosaccharide residue. The precipitating agent can be any acidic salt that provides a halo counterion, for example, HCl, HBr, HI. Preferably, the counterion is a chloride ion. The aliphatic polyamine includes polyamines such as triethylene tetramine, diethylene triamine, ethylene diamine, and diaminopropane.
The saccharide of interest can be a monosaccharide having 4 to 7 carbon atoms or an oligosaccharide consisting of any two or more monosaccharides. Some examples of monosaccharides include glucose, galactose, fucose, or allose. The oligosaccharide may, as described supra, consist of any two or more monosaccharides joined through one or more glycosidic linkages.
When each of the saccharide residues, R1 and R2 is a monosaccharide residue, it must be the same monosaccharide. When each of the saccharide residues, R1 and R2, is an oligosaccharide residue, the saccharide unit that forms the N-glycosyl bond with the amine of the polyamine linker must be the same for each oligosaccharide residue. Alternatively, one of R1 or R2 can be a monosaccharyl and the other can be an oligosaccharyl, provided that, in case of the oligosaccharide, the saccharide unit that forms the N-glycosyl bond with the amine of the polyamine linker must be the same as the monosaccharide.
For example, if R1 is an oligosaccharide that consists of glucose-mannose-allose residues and it is the allose that is involved in glycosidic linkage with the amino group of the polyamine linker, then R2 must be an allose residue or the glycosidic-bond forming saccharide unit of an oligosaccharide. Additionally, the group whose conformation at the C2 position is being determined should preferably be the same on each saccharide residue that is involved in glycosidic linkage with the amino group of the polyamine linker.
Optionally, the method can be automated either to prepare the chloride salt of the glycosylated polyamine or to observe the presence or absence of the precipitate. A variety of methods can be used to observe the chloride precipitate, and include calorimetric, and spectrophotometric methods. The observation of the precipitate, either manually or through automation, is within the capability of one of ordinary skill in the art.
Some specific examples where an equatorial conformation at the C2 position of a saccharide were determined include glucose, galactose, allose or fucose. One specific example where an axial conformation at the C2 position was determined is in the case of mannose. Additional examples can include oligosaccharides wherein the sugar moiety forming the N-glycosidic linkage with the polyamine is a hexose, such as, glucose, galactose, allose, fucose, or mannose.